This invention relates to an electrophoresis cassette useful for conducting gel electrophoresis separations.
Electrophoresis is the resolution of a complex mixture of macromolecules on the basis of charge and/or size under the influence of an electric field and is a primary tool in biochemistry, used to separate complex mixtures of molecules such as proteins or nucleic acids into their individual components. Electrophoretic analysis is based upon the fact that each molecule is characterized by a particular electrophoretic mobility under a given set of conditions. Macromolecules will migrate within a voltage gradient according to their net charge and will reach equilibrium at their isoelectric point at which their net mobility will be zero. For example, many proteins exhibit a net negative charge which is affected by the surrounding pH. When a mixture of proteins is placed in a support medium, such as a buffered gel, which is subjected to a voltage gradient, each component is caused to migrate through the support medium at its characteristic rate for that set of conditions. Electrophoretic mobility is a function of other factors which are controlled by experimental conditions.
It is common practice to conduct electrophoresis in a buffered gel positioned between two flat plates, usually transparent glass or plastic and separators between the plates which provide essential support for the gel. In order to provide accurate sample resolution, it is necessary that the gel composition be uniform and that the gel thickness be uniform. These conditions are necessary in order to avoid factors which affect molecular electrophoretic mobility other than the characteristics of the molecules being separated. In use, the cassette is positioned between two buffer solutions after the sample or samples have been placed on one gel surface. A voltage is applied between the buffers which causes the samples to migrate within the gel. Upon completion of sample preparation, the gel is separated from the plates for analysis.
Presently, a cassette is produced wherein a void volume is formed between two plates separated by two separators. A suitable separation gel medium such as agarose or a polyacrylamide is poured, in liquid form, into the void volume and allowed to polymerize therein. During formation of the gel, the two plates are compressed to the separators to prevent leakage of the gel material from the void volume and to assure a uniform distance between the plates, which, in turn, assures a uniform gel thickness.
In order to form sample wells at a top end of the cassette, a removable piece having fingers having the desired shape of the wells is positioned at the top end so that the fingers extend into the separation medium while it is polymerizing. After the gel is formed, the fingers are removed from the gel to leave wells wherein samples can be positioned. The use of this removable piece is undesirable since an additional apparatus is required and its use requires an additional manipulative step which must be conducted with care in order to avoid ripping the gel.
Accordingly, it would be desirable to provide a means for forming wells in the gel portion of a cassette which eliminates the need for a piece which must be removed subsequent to gel formation. In addition, it would be desirable to provide such a means which provides a uniform electrical resistance or field so that separation of samples can be maintained during gel electrophoresis.